This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of the grant is to define the function of a family of immune evasion proteins found in gamma-2 herpesviruses. Aim 1: Molecular mechanism of MHC class I downregulation by KSHV. To examine the molecular mechanism by which the ORFs K3 and K5 down-regulate cell surface immunoreceptors such as MHC I, we proposed to study their function both in KSHV-infected cells as well as in K3- and K5-transfected cells. Mechanistic aspects such as the role of ubiquitination as well as structural requirements within K3 and K5 responsible for substrate and species specificity will be dissected. Aim 2: Identification of cellular proteins interacting with K3 and K5. K3 and K5 contain a zinc-finger motif known as the PHD/LAP-finger that is related in sequence and structure to the RING domain found in many ubiquitin ligases. We proposed to examine whether known or unknown cellular proteins interact with K3 and K5 using co-immunoprecipitation and yeast-two hybrid assays. We also wanted to reconstitute the K3 and K5 function as ubiquitin ligase in vitro. Aim 3: Function of K3 and K5 for immune evasion in vivo Since KSHV cannot be studied in animals, we suggested using MHV68 as a surrogate model by replacing the endogenous K3 of MHV68 with KSHV- K3 and K5 and testing the resulting chimeric virus in mice.